Throughout this application, various references are cited in parentheses to describe more fully the state of the art to which this invention pertains. Full bibliographic information for each citation is found at the end of the specification, immediately preceding the claims. The disclosure of these references are hereby incorporated by reference into the present disclosure.
The ability of cancer cells to evade or escape immune detection and destruction is recognized as a key hallmark of carcinogenesis and cancer progression (Rodriguez, P. C., A. H. Zea, and A. C. Ochoa. 2003. Mechanisms of tumor evasion from the immune response. Cancer Chemother Biol Response Modif 21:351.). Essential to this process is the ability of tumor cells to create a state of localized immune tolerance or non-responsiveness towards their otherwise immunogenic antigens. Such immunosuppression and evasion can be achieved through a variety of mechanisms at the disposal of cancerous cells, many of which have been characterized; secretion of Th2-associated cytokines such as IL-10 or TGF-β leading towards Th2 polarization (Sheu, B. C., R. H. Lin, H. C. Lien, H. N. Ho, S. M. Hsu, and S. C. Huang. 2001. Predominant Th2/Tc2 polarity of tumor-infiltrating lymphocytes in human cervical cancer. J Immunol 167:2972.), over-expression of Fas-L/TRAIL 15,16, over-expression of complement inhibitors (DAF, CD55) (Murray, K. P., S. Mathure, R. Kaul, S. Khan, L. F. Carson, L. B. Twiggs, M. G. Martens, and A. Kaul. 2000. Expression of complement regulatory proteins-CD 35, CD 46, CD 55, and CD 59-in benign and malignant endometrial tissue. Gynecol Oncol 76:176.) 21 and over-expression of HLA-G protecting against NK-induced lysis (Ugurel, S., Reinhold, U. & Tilgen, W. HLA-G in melanoma: A new strategy to escape from immunosurveillance? Onkologie 25, 129-34 (2002).
An immunosuppressive enzyme in this process of malignant tolerance has been identified as indoleamine 2,3-dioxygenase (IDO), a tryptophan catabolizing enzyme (Uyttenhove, C., L. Pilotte, I. Theate, V. Stroobant, D. Colau, N. Parmentier, T. Boon, and B. J. Van den Eynde. 2003. Evidence for a tumoral immune resistance mechanism based on tryptophan degradation by indoleamine 2,3-dioxygenase. Nat Med 9:1269). IDO is the rate-limiting enzyme in the kynurenine pathway which converts the essential amino acid L-tryptophan to L-kynurenine. Through the degradation of tryptophan, IDO is believed to suppress T cells through two primary mechanisms: 1) through the depletion of tryptophan (tryptophan depletion theory) and 2) through the generation of toxic downstream metabolites of the kynurenine pathway (ie. quinolinic and picolinic acid) which are known to induce T cell apoptosis (tryptophan utilization theory)(Moffett, J. R., and M. A. Namboodiri. 2003. Tryptophan and the immune response. Immunol Cell Biol 81:247).
IDO is expressed by a variety of antigen presenting cells (APCs) including monocyte-derived macrophages, dendritic cells, and many different subsets of tumor cells both murine and human. Expression of IDO by both professional APCs (monocyte-derived macrophages, human and murine dendritic cells) and nonprofessional APCs (tumor cell lines) has been proven to inhibit locally responding T cells in vitro (Bauer, T. M., L. P. Jiga, J. J. Chuang, M. Randazzo, G. Opelz, and P. Terness. 2005. Studying the immunosuppressive role of indoleamine 2,3-dioxygenase: tryptophan metabolites suppress rat allogeneic T-cell responses in vitro and in vivo. Transpl Int 18:95; Mellor, A. L., D. B. Keskin, T. Johnson, P. Chandler, and D. H. Munn. 2002. Cells expressing indoleamine 2,3-dioxygenase inhibit T cell responses. J Immunol 168:3771). Expression of IDO has also been implicated in the generation of tryptophan metabolites such as quinolinic acid, 3-hydroxyanthranilic acid, anthranilic acid, kynurenine, and 3-hydroxykynurenine, which are considered toxic and can induce apoptosis of locally responding T cells (Moffett, J. R., and M. A. Namboodiri. 2003. Tryptophan and the immune response. Immunol Cell Biol 81:247; Fallarino, F., U. Grohmann, C. Vacca, R. Bianchi, C. Orabona, A. Spreca, M. C. Fioretti, and P. Puccetti. 2002. T cell apoptosis by tryptophan catabolism. Cell Death Differ 9:1069; Frumento, G., R. Rotondo, M. Tonetti, G. Damonte, U. Benatti, and G. B. Ferrara. 2002. Tryptophan-derived catabolites are responsible for inhibition of T and natural killer cell proliferation induced by indoleamine 2,3-dioxygenase. J Exp Med 196:459; Terness, P., T. M. Bauer, L. Rose, C. Dufter, A. Watzlik, H. Simon, and G. Opelz. 2002. Inhibition of allogeneic T cell proliferation by indoleamine 2,3-dioxygenase-expressing dendritic cells: mediation of suppression by tryptophan metabolites. J Exp Med 196:447.
The involvement of IDO in tumor evasion has been implicated in murine models. For example, P815 murine mastocytoma cells transfected with IDO were able to form large and stable tumors when introduced into pre-immunized hosts which would normally reject the tumors outright (Uyttenhove, C., L. Pilotte, I. Theate, V. Stroobant, D. Colau, N. Parmentier, T. Boon, and B. J. Van den Eynde. 2003. Evidence for a tumoral immune resistance mechanism based on tryptophan degradation by indoleamine 2,3-dioxygenase. Nat Med 9:1269). The immunosuppressive effect of IDO was completely reversed by the introduction of 1-MT (a chemical with potential toxic effects) leading to renewed tumor rejection.
Due to its ability to suppress immune responses towards endogenous tumor antigens, IDO as well as other immunosuppressive molecules represent ideal targets for immunomodulatory drugs which are used to leverage the efficacy of standard chemotherapeutic agents. In the case of IDO, currently used immunomodulatory drugs pose potential toxic threats towards humans especially if required in large doses.
RNA interference (RNAi) refers to a process of sequence-specific post-transcriptional gene silencing in animals mediated by short interfering RNAs (siRNAs). There exist several different methods for inhibiting gene expression using RNAi such as described for example in WO 02/055692, WO 02/055693, EP 1144623 B1 and WO 03/074654. WO 02/08644 and WO 04/048938 suggest that siRNA technology may be used for inhibiting cancer, however, no studies were demonstrated to support such a contention.
The ability to inhibit IDO as well as inhibit other immunosuppressive tumor derived molecules without the use of detrimental chemicals is desirous. The Applicant has therefore provided a method utilizing siRNA by which tumor derived immunosuppression can be inhibited in a manner that obviates one of more deficiencies of current therapies that may debilitate the immune system and potentially pose damaging and toxic effects upon the body.